Restrictocin is a fungal cytotoxin which cleaves RNA in intact ribosomes at a unique site in the "sarcin loop." It also has the ability to cross membranes to enter cells. No structure is currently available and, at 17 kilodaltons molecular weight (148 amino acids), it is a good candidate for structure determination using multinuclear, multidimensional NMR methods. The initial steps in this undertaking will be to determine the pKa values of the eight histidines of restrictocin using one- and two-dimensional 1H NMR methods (including DQF-COSY). Also included will be studies of a mutant, H136L, which lacks the histidine in the putative active site region; this will provide a direct assignment of the histidine-136 resonances. With 15N-labeled protein, three-dimensional 1H-15N NMR experiments (e.g., HMQC-NOESY) will be performed for sequential assignment purposes. Subsequently, fully 13C-15N labeled protein will be prepared and used for the complete structure determination.